The initial outbreak of West Nile virus (WNV) in the Western Hemisphere occurred in New York in 1999, and since then the virus has rapidly spread across the United States and Canada, where it has been responsible for significant human morbidity and mortality. In contrast, there have been no human cases of WNV in the Yucatan Peninsula (YP) of Mexico, nor have there been any reports of widespread WNV associated illness in horses or birds in this region, despite evidence of WNV activity. Indeed, we and our Mexican colleagues at the Universidad Autonoma de Yucatan (DAY) in Merida detected antibodies to WNV in horses, birds and other vertebrate animals in the YP. Most pertinent to this proposal, however, are the findings from a serosurvey conducted on Cozumel Island; antibodies to WNV were detected in 75 of 144 (52%) horses, but none of the horses had signs of illness and none had a history of WNV-like illness. Thus, the underlying hypothesis in this proposal is that the dominant WNV variants in the YP are genotypically and phenotypically distinct from the dominant WNV variants in the U.S. In this grant, we and our colleagues at the UAY will obtain WNV isolates in the YP, and define their genotypes and cell culture phenotypes. In specific aim 1, mosquitoes will be trapped at sites in the YP where seropositive vertebrates have previously been identified. Mosquitoes will be assayed for WNV by RT-PCR and virus isolation. These studies will identify likely vectors of WNV in the YP, thereby providing the groundwork for future vector competence and host-feeding behavior studies. In specific aim 2, WNV isolates will be examined by partial nucleotide sequencing and phylogenetic analysis. Dominant WNV variants will be completely sequenced. In specific aim 3, the cell culture phenotypes (i.e. replication kinetics and yields, plaque morphologies and temperature sensitivities) of dominant WNV variants from the YP and U.S. will be compared. These studies will provide the groundwork for future in vivo pathogenesis studies in vertebrate hosts. RELEVANCE: In this proposal, the genotypes and cell culture phenotypes of dominant WNV variants in the YP will be characterized, and likely vectors of WNV in this region will be identified. This information will help determine why there has not been an epidemic of WNV in the YP, and will identify risk factors associated with WNV disease. [unreadable] [unreadable] [unreadable]